Background: Few studies have investigated the effects of adopting a specific and prolonged posture on cyclists. This study aimed to evaluate the upright spine in a sample of recreational cyclists and compare it with a sample of non-cyclists, though still athletes, through a 3D scanning method. Methods: Forty-eight participants were enrolled in this observational study. The sample consisted of 25 cyclists for the cycling group and 23 non-cyclist athletes for the control group. The Spine3D device (Sensor Medica, Guidonia Montecelio, Rome, Italy) was used to evaluate the spine of the participants in both groups. Results: The results showed significantly greater spine inclination in the cycling group compared to the control group (p < 0.01). Furthermore, there was a significant decrease in lumbar lordosis in the cycling group compared to the control group (p < 0.01). Conclusions: This case–control study raises the possibility that the onset of lower back pain in cyclists may be due to a reduction in lumbar lordosis. Furthermore, this study demonstrated that the Spine3D device can be used in sports to monitor the spine of athletes to prevent and reduce musculoskeletal deficits.